ABSTRACT Recent studies have demonstrated that memories are formed and expressed from small groups of neurons, neural ensembles, within brain areas pertinent to the memory. Repeated drug use establishes drug-related memories thought to drive relapse. When these memories are recalled by re-exposure to drug-associated cues, context, or the drug itself, they become labile for several hours and are then reconsolidated to maintain or strengthen them. However, they can also be disrupted with certain amnestic agents or behavioral manipulations given shortly after recall. We have shown that reconsolidation of cocaine memories can be disrupted by manipulating the prelimbic portion of the rat medial prefrontal cortex (PL PFC). PL PFC neurons projecting to the nucleus accumbens (NAc) critically control reinstatement, the rodent model for relapse in humans. In our Preliminary Studies, this memory disruption may depend on updating the memory during a memory reactivation session so that it becomes labile for disruption. Updating a well-trained (habit) memory, such as that learned during cocaine self-administration in rats, appears to depend on creating prediction error; that is, a difference in what the rat expects to receive by pressing a lever and what it actually receives after pressing the lever to receive cocaine. Our Preliminary Studies show if we use the same reinforcement schedule as given during the many self-administration training days, a fixed ratio 1 (FR1), the memory may not become labile for disruption, but that a different reinforcement schedule, a variable ratio 5 (VR5) that is unpredictable, may allow for the memory to be disrupted. However, we do not know which ensembles of neurons in the PL PFC allow these memories to become labile for disruption. Here we propose to use in rats a new, highly sensitive robust activity marking (RAM) system to identify neural ensembles in the PL PFC activated by two types of memory reactivation sessions, one that involves memory updating and the other that does not. Specifically, we will 1) identify which neural ensembles are activated within the PL PFC during cocaine memory reactivation in cocaine self-administering rats, 2) identify the phenotype of these ensembles, and 3) begin to define the neural circuitry involved in memory updating. We will use dual viral injections, behavioral, and immunohistochemical approaches. This proposal is significant because it will allow us to decipher the underlying neural ensembles that render difficult-to-disrupt cocaine memories labile for disruption, with the long-term goal of reducing relapse to cocaine.